The invention relates to reference electrodes for use in voltammetric measuring chains for oxidative as well as reductive detection of analytes, which are open with respect to the measuring medium. A metal-containing phthalocyanine or a metalloporphyrin is employed as potential-determining electrode material for reference electrodes of the invention used in oxidative detection. A fourth period metal, preferably copper, and a salt of said metal is employed as potential-determining electrode material for reference electrodes of the invention used in reductive detection. The potential-determining electrode material is coated as a layer or component of a layer on a planar support. The open reference electrodes according to the invention are particularly suited for voltammetric chemo- or biosensors having a planar structure.
When utilizing mass production technologies for voltammetric chemo- and biosensors as known from thin and thick film technologies in microelectronics, problems arise in transferring to planar structures, particularly in case of conventional reference electrodes.
In general, a measuring cell consisting of a measuring or working electrode and a reference electrode, or a potentiostatically working measuring cell consisting of a working electrode, a reference electrode and an auxiliary electrode or counterelectrode is used in the voltammetric detection of analyte concentrations in solution, where the electrodes submerge in the solution to be measured. To ensure a constant reference potential which is required in all types of voltammetric detections in order to adjust a defined working electrode polarization potential, reference electrodes are used that are closed with respect to the measuring medium. A typical example of a voltammetric sensor is the ampero-metric Clark type oxygen measuring cell which consists of a polarizable working electrode and a non-polarizable reference electrode. When applying a suitable polarization potential between the two electrodes, oxygen will be reduced electro-chemically at the working electrode, thus generating an analyte-proportional depolarization current between the two electrodes in the external circuit. Particularly in the event of current-carrying reference electrodes, care must be taken to have a sufficiently large surface, which must be a multiple of the working electrode surface in order to maintain a constant reference potential. Alternatively, the so-called potentiostate principle is used wherein the reference electrode is virtually free of current load as a result of a suitable electronic control and the use of an additional auxiliary electrode (counterelectrode).
Being electrodes of the second type, a common feature of all traditional reference electrodes for use in aqueous media is to consist of a metal (e.g., mercury or silver), a sparingly soluble compound of said metal, and a phase containing the anion of the metal compound. Consequently, the potential-determining reference electrode material is surrounded by a defined anion concentration of said metal compound and connected with the working electrode via an ionically conducting salt bridge which has a diaphragm at its end.
The salt bridge, normally being filled with chloride concentrations of between 1 M and 3 M, and the diaphragm providing ionic contact with the solution to be measuredxe2x80x94in a way that mixing with the salt bridge electrolyte cannot take placexe2x80x94ensure a constant ratio of concentrations of metal/metal salt and metal salt anions and thus, a constant reference potential.
Transferring such a closed reference electrode system to planar structures is only possible with great effort, thus impeding cost-effective mass production. Therefore, voltammetric measuring chains of planar structure, particularly for cost-effective production of voltammetric chemo- and bio-sensors based on thin and thick film technologies, frequently work with open reference electrodes, i.e., in a way that the reference electrode surface is directly exposed to the measuring medium.
Preferably, silver/silver halides are used as metal/metal salt reference electrode system (EP 0,304,933; DE 33 09 251; U.S. Pat. No. 5,509,410). An approximately constant concentration of silver ions is required to maintain the equilibrium potential of a silver reference electrode, which is why silver salts sparingly soluble in water, such as silver chloride, are used. Due to the solubility product, the concentration of silver ions in aqueous media depends substantially on the concentration of chloride ions. Now, when using the silver/silver chloride system without a salt bridge, i.e., with direct exposure to the measuring medium, a number of interferences generally occur, particularly in those cases where measurements have to be performed in undefined media as represented by actual samples. For example, the steady state ionic concentration at the metal/metal salt boundary surface undergoes changes depending on the flow velocity, resulting in a short-term interference of the equilibrium potential and thus, in a potential drift. Moreover, anions of unknown composition and undefined concentration are present in the measuring medium, which in turn produce silver salts and may dominate the reference potential as a result of their solubility product. At least, however, they may give rise to a mixed potential resulting in a more or less substantial potential drift and conceivably, in a high transition resistance Hence, the composition of the ions and their concentration in the measuring medium may have a substantial effect on the reference potential. As described above, an open reference electrode system also is biased by adsorbent substances, metal chelating agents or electrically active substances in the measuring medium and ultimately, by the conditions of diffusion and/or convection at the electrode surface. This situation can largely be compensated by diluting the sample with a defined electrolyte solution and maintaining constant flow conditions.
Under specific conditions, however, particularly as encountered in amperometric biosensors where the biocomponent requires specific buffer materials or ions for a stable and sensitive indication reaction, considerable shifts of the reference potential may occur. For example, when using a phosphate buffer in an open silver/silver chloride reference electrode, silver phosphate will form on the silver electrode despite the presence of chloride ions, giving rise to a reference electrode potential shift by about 150 mV to the negative side. Thus, compared to a closed silver/silver chloride reference electrode, a polarization potential more negative by about 150 mV has to be applied to the working electrode in a reductive indication of an analyte present in its oxidized form, which ultimately also involves an increased susceptibility to interference in the measuring chain with open reference system.
Another aspect of general importance for the oxidative indication of e.g. hydrogen peroxide formed in oxidase-catalyzed reactions relates to the comparatively high polarization potential required in a conventional measuring chain array comprising a platinum working electrode and a silver/silver chloride reference electrode. While the use of an open measuring chain in phosphate buffer implies a reduction of the polarization potential required, the polarization potential to be appliedxe2x80x94still being from +450 mV to +500 mVxe2x80x94involves a considerable risk of interference current effects. Moreover, in case of a quasi-continuous inflow of such an open measuring chain, the transient action of the measuring chain during the transition between resting and flowing medium is expected to proceed slowly as a result of the change in ion concentration and the associated change of the equilibrium potential between metal and metal salt, which in the worst case overlays the generation of measuring values.
Especially in miniaturized potentiometric sensor arrays, attempts were made to adapt the situation of a conventional reference electrode structure in such a fashion that polymeric materials or diaphragms as diffusion barrier layers containing the metal salt anion were coated layer by layer on the reference electrode material (EP 0,247,535; DE 195 33 059; DE 195 34 925), or appropriate reservoirs containing reference electrolyte were provided (Lambrechts, M., Sansen W.: Biosensors: Microelectrochemical devices, Institute of Physics Publishing, Bristol Philadelphia and New York, 1992). In a similar type of construction, planar reference electrodes for amperometric sensors are known (DE 42 41 206; U.S. Pat. No. 4,980,043). Both of these approaches have in common that the layers, diffusion barriers or miniaturized reservoirs ensure not more than limited retaining of the reference electrolyte, and leaking of electrolyte into the measuring medium or diffusion of the solution to be measured to the reference system cannot be prevented over a prolonged period of operation.
The patent specification DE 43 02 322 suggests the use of perchlorate as potential-determining ion in combination with a perchlorate-sensitive diaphragm between the reference electrolyte reservoir and the measuring medium. By using e.g. potassium perchlorate which is introduced in a solid form into the reservoir and has a substantially lower solubility in water than reference electrolytes of second type electrodes, a substantially improved long-term stability is achieved in combination with the perchlorate-sensitive diaphragm. Such a reference system is also useful in potentiostatically operated voltammetric sensors, butxe2x80x94like all the variants including a reservoirxe2x80x94requires a three-dimensional sensor design.
In addition to using a redox reference electrode, another, comparatively expensive solution (DE 43 02 323) suggests a so-called protective electrode within the space of the reference electrolyte, spatially situated between the diaphragm and reference electrode. The potential is measured between the two electrodes. In case of a potential difference at the protective electrode with respect to the reference electrode as a result of inwardly diffusing electrically active substances, a current is generated via an additional counterelectrode in analogy to the potentiostate principle, which reduces or oxidizes the interfering substances until potential parity between protective and reference electrodes is re-established.
In amperometric enzyme sensors, alternative reference electrode materials such as silver/palladium mixtures (U.S. Pat. No. 5,820,551), stainless steel alloys (U.S. Pat. No. 5,736,029) and graphite (EP 0,776,675; U.S. Pat. No. 5,916,156) have been used in two-electrode systems as pseudo-reference electrodes.
In three-electrode arrays on xe2x80x9copenxe2x80x9d planar sensor structures operated in so-called potentiostatic operation, silver/silver chloride has essentially been used as reference electrode material until now, involving the problems described above.
The object of the invention is to provide voltammetric reference electrodes that would avoid the above-mentioned drawbacks and permit less trouble-prone, correct and reproducible measurements. In particular, these electrodes should be cost-effective in production and capable of miniaturization.
Surprisingly, metal-containing phthalocyanines or metalloporphyrins, each present in homogeneous mixture with an inert, conductive material or with an inert, conductive material and a curable binder, were found to be particularly suitable as potential-determining electrode material coated on a planar support and to be used for an open reference electrode of a voltammetric measuring chain, at the working electrode of which an oxidative detection of redox-active analytes takes place. Carbon, e.g. graphite or pyrolytic carbons such as carbon black, or glass-carbon ground to powder, or noble metal powders of gold, platinum or palladium, preferably carbon or a mixture of various types of carbon are used as inert, conductive material. Polymers, preferably poly(vinyl chloride), silicone resins, epoxide resins, phenol resins, acrylates, or mixtures thereof can be used as binders.